共查询到20条相似文献,搜索用时 9 毫秒
1.
Purification and characterization of endo-β-1,3-1,4-d-glucanase activity from Bacillus licheniformis
Jorge Lloberas Enrique Querol Jordi Bernués 《Applied microbiology and biotechnology》1988,29(1):32-38
Summary An extracellular -glucanase from Bacillus licheniformis has been isolated and characterized. Isolation has been performed by salting out and gel filtration chromatography, yielding a homogeneous active component with a molecular mass of 27 000–28 000 daltons and an isoelectric point of 4.7. In addition to being quite a thermostable protein (optimal temperature 55°C) the enzyme is active under a wide range of conditions including pH (4.0–10.5), and in the presence of a large number of metal ions, sodium dodecylsulphate and ethylenediaminetetraacetate. The simple purification procedure and useful properties make this enzyme suitable for brewing processes. 相似文献
2.
Two exo-β-glucanases (LP-ExoI, 83 kDa and LP-ExoII, 71 kDa) were extracted and partially purified from the cell wall of Lilium longiflorum pollen tubes. Both LP-ExoI and LP-ExoII hydrolyzed laminarin (1,3-β-glucan). These enzymes also exhibited some activity toward
1,3:1,4-β-glucans of Hordeum vulgare and Cetraria islandica and the 1,6-β-glucan of Umbilicaria papullosa. The pH for optimum activity for both exo-β-glucanases was 5.5. Methylation analysis of the reaction products revealed that
purified LP-ExoI decreased both 1,3- and 1,4-glucosyl linkages in hemicellulosic polysaccharides isolated from the cell wall
of lily pollen tubes. D-gluconolactone and nojirimycin, inhibitors of glucosidase, inhibited activities of both exo-β-glucanases,
as well as growth of the lily pollen tubes. These results disclosed that the wall-bound exo-β-glucanases play an important
role in the regulation of lily pollen tube growth.
Received: 3 January 2000 / Revision accepted: 8 March 2000 相似文献
3.
Pengjun Shi Guoyu Yao Peilong Yang Ning Li Huiying Luo Yingguo Bai Yaru Wang Bin Yao 《Applied microbiology and biotechnology》2010,85(5):1483-1490
An endo-1,3-β-d-glucanase gene, designated as bglS27, was cloned from Streptomyces sp. S27 and successfully expressed in Escherichia coli BL21 (DE3). The full-length gene contains 1,362 bp and encodes a protein of 453 amino acids with a calculated molecular mass
of 42.7 kDa. The encoded protein comprises a catalytic module of glycosyl hydrolase family 16, a short glycine linker region,
and a family 13 carbohydrate-binding module. The purified recombinant enzyme (BglS27) showed optimal activity at 65°C and
pH 5.5 and preferentially catalyzed the hydrolysis of glucans with a β-1,3-linkage using an endolytic mode of action. The
specific activity and K
m value of BglS27 for laminarin were 236.0 U mg–1 and 1.89 mg ml–1, respectively. In antifungal assay, BglS27 had the ability to inhibit the growth of phytopathogenic fungi Rhizoctonic solani and Fusarium oxysporum and some mycotoxin-producing fungi Fusarium crookwellense and Paecilomyces variotii. These favorable properties make BglS27 a good candidate for utilization in biotechnological applications such as plant protection,
feed, and food preservation. 相似文献
4.
可变剪接是基因表达调控的一种重要方式。本研究利用RT-PCR克隆鉴定到草菇exo-β-1,3-葡聚糖酶基因(exg2)的4种可变剪接体(exg2V1,exg2V2,exg2V3和exg2V4),其中,exg2V1第7个内含子保留;exg2V2第11个内含子保留同时剪切掉第12个外显子的后58bp;exg2V3同时保留第7个和第17个内含子;exg2V4同时保留第2、第9和第17个内含子。4种剪接体均在可变剪接位点提前出现终止密码子,导致预测编码的氨基酸序列中包含不完整的结构域。定量PCR结果显示:草菇5个发育时期中,4种可变剪接体表达量均比标准剪接体exg2低,但4种可变剪接体的表达也表现出一定的时期和组织差异性。初步推断发现的草菇exg2基因4种可变剪接体均为无义介导的mRNA降解,通过此方式达到对exg2基因标准剪接转录本的精准调控。 相似文献
5.
Zakharenko AM Kusaykin MI Kovalchuk SN Anastyuk SD Ly BM Sova VV Rasskazov VA Zvyagintseva TN 《Carbohydrate research》2011,346(2):243-252
The retaining endo-1,3-β-d-glucanase (EC 3.2.1.39) was isolated from the crystalline styles of the commercially available Vietnamese edible mussel Perna viridis. It catalyzes hydrolysis of β-1,3-bonds in glucans and enables to catalyze a transglycosylation reaction. Resources of mass-spectrometry for analysis of enzymatic products were studied. cDNA sequence of endo-1,3-β-d-glucanase was determined by RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) methods. The cDNA of 1380bp contains an open reading frame of 1332bp encoding a mature protein of 328 amino acids. On basis of amino acid sequence analysis endo-1,3-β-d-glucanase was classified as a glycoside hydrolase of family 16. 相似文献
6.
Ying Peng Zhen-Ming Chi Xiang-Hong Wang Jing Li 《Applied microbiology and biotechnology》2009,85(1):85-94
The extracellular β-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific β-1,3-glucanase activity as compared to that in the
supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from
sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa.
The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The
optimal pH and temperature of the purified enzyme were 4.0 and 40°C, respectively. The enzyme was significantly stimulated
by Li+, Ni2+, and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol
bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1,10-phenanthroline. The K
m and V
max values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser
desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR
which is the conserved motif of the β-1,3-glucanases from other yeasts. 相似文献
7.
8.
1,3-β-Glucan synthase activity has been demonstrated in particulate fractions of bark extracts from Mexican lime. With respect to substrate, the enzyme kinetics did not conform to the Michaelis-Menten equation. The value of the Hill coefficient was 1.2 and S0.5 is 1.1 mM. The enzyme had an optimum pH of 7.5. Maltose, sucrose, and especially cellobiose and glucose, were enzyme activators when tested at physiological concentrations. In the presence of 15 mM MgCl2 the enzymic activity was stimulated at 10 μM UDP-glucose but decreased at 1 mM UDP-glucose, suggesting a minor 1,4-β-glucan synthase activity. 相似文献
9.
Huang JW Cheng YS Ko TP Lin CY Lai HL Chen CC Ma Y Zheng Y Huang CH Zou P Liu JR Guo RT 《Applied microbiology and biotechnology》2012,94(1):111-121
1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ~30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity. 相似文献
10.
《Carbohydrate research》1987,166(1):145-155
An enzyme active against O-(carboxymethyl)cellulose (CMC) was purified from a synthetic medium containing ball-milled cellulose wherein Ruminococcus albus had been cultivated for 70 h. After 570-fold purification, a homogeneous enzyme was obtained in a yield of 3%. The enzyme degraded CMC (molecular weight, 180,000; degree of substitution, 0.6) to a smaller polymer having a molecular weight of ∼20,000, and generated a small proportion of glucose, but negligible proportions of such cello-saccharides as cellobiose, cellotriose, cellotetraose, or cellopentaose. The fact that the enzyme could produce water-insoluble fragments was discovered by dissolving substrate and products in Cadoxen solution. No water-soluble cello-oligomers were detected by thin-layer chromatography after degradation of water-insoluble cellulose by the purified enzyme. Therefore, the enzyme was classified as an endo-(1→4)-β-d-glucanase. 相似文献
11.
Molecular-sieve chromatography of an extract from ungerminated rye indicated the presence of enzymes which hydrolysed cellobiose, laminaribiose and the β-glucans cellodextrin, laminarin and barley β-glucan. A purified endo-β-1,3-glucanase was prepared from the extract by ammonium sulphate fractionation and molecular-sieve chromatography on Biogel P60. The substrate specificity and some properties of the enzyme are reported and the in vivo role of the enzyme is discussed. 相似文献
12.
《Carbohydrate research》1986,153(2):271-283
The ability of imidates, thioimidates, and dithioates to react with o-aminophenol (2) and 5,6-diamino-1,3-dimethyluracil (6) was studied, using non-saccharide model compounds, as well as saccharide derivatives. All of the model compounds gave 2-methylbenzoxazole, but only ethyl dithioacetate gave a purine derivative with 6. Methyl 2,5-anhydro-d-allonoimidate hydrochloride reacted with 2 to yield 2-β-d-ribofuranosylbenzoxazole, but failed to react with compound 6. On reaction with compound 6 such fully acylated thioimidates as ethyl and benzyl 2,5-anhydrotri-O-benzoyl- or tri-O-p-toluoyl-d-allonothiomidate hydrochloride yielded amidines that underwent aromatization of the furanose ring. Such monoacylated thioimidates as ethyl or benzyl 2,5-anhydro-6-O-benzoyl--d-allonothioimidate hydrochloride yielded, with compound 6, 8-(5-O-benzoyl-β-d-ribofuranosyl)-1,3-dimethylxanthine, without aromatization. Such dithioates as benzyl 2,5-anhydro-6-O-benzoyl-d-allonodithioate and ethyl 2,5-anhydrotri-O-benzoyl-d-allonodithioate were obtained by treating the corresponding thioimidate with H2S in pyridine. With compound 6, the first yielded 8-(5-O-benzoyl-β-d-ribofuranosyl)-1,3-dimethylxanthine, which afforded the free C-nucleoside 1,3-dimethyl-8-β-d-ribofuranosylxanthine on treatment with methanolic ammonia. 相似文献
13.
Following the discovery of an exo-1,3/1,4-β-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 ? resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue β-d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards β-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a β-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 3UT0, 3USZ, 3F95 and 3RRX. 相似文献
14.
Monzani PS Bressan FF Mesquita LG Sangalli JR Meirelles FV 《Genetics and molecular research : GMR》2011,10(2):604-614
Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed β-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the β-casein promoter, independent of β-casein expression. 相似文献
15.
Oscar Goñi 《Phytochemistry》2011,72(9):844-854
A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO2-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a Mr of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low kcat (3.10 ± 0.04 s−1) and Km (0.32 ± 0.03 mg ml−1) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high kcat at both 37 and 5 °C, and a low Ea (6.99 kJ mol−1) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD50 was 8.7 μg ml−1 (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO2-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO2. 相似文献
16.
Tatsuji Sakamoto Yuichi Nishimura Yosuke Makino Yoichi Sunagawa Naoki Harada 《Applied microbiology and biotechnology》2013,97(7):2895-2906
An endo-β-1,4-galactanase (PcGAL1) and an exo-β-1,4-galactanase (PcGALX35C) were purified from the culture filtrate of Penicillium chrysogenum 31B. Pcgal1 and Pcgalx35C cDNAs encoding PcGAL1 and PcGALX35C were isolated by in vitro cloning. The deduced amino acid sequences of PcGAL1 and PcGALX35C are highly similar to a putative endo-β-1,4-galactanase of Aspergillus terreus (70 % amino acid identity) and a putative β-galactosidase of Neosartorya fischeri (72 %), respectively. Pfam analysis revealed a “Glyco_hydro_53” domain in PcGAL1. PcGALX35C is composed of five distinct domains including “Glyco_hydro_35,” “BetaGal_dom2,” “BetaGal_dom3,” and two “BetaGal_dom4_5” domains. Recombinant enzymes (rPcGAL1 and rPcGALX35C) expressed in Escherichia coli and Pichia pastoris, respectively, were active against lupin galactan. The reaction products of lupin galactan revealed that rPcGAL1 cleaved the substrate in an endo manner. The enzyme accumulated galactose and galactobiose as the main products. The smallest substrate for rPcGAL1 was β-1,4-galactotriose. On the other hand, rPcGALX35C released only galactose from lupin galactan throughout the reaction, indicating that it is an exo-β-1,4-galactanase. rPcGALX35C was active on both β-1,4-galactobiose and triose, but not on lactose, β-1,3- or β-1,6-galactooligosaccharides even after 24 h of incubation. To our knowledge, this is the first report of a gene encoding a microbial exo-β-1,4-galactanase. rPcGAL1 and rPcGALX35C acted synergistically in the degradation of lupin galactan and soybean arabinogalactan. Lupin galactan was almost completely degraded to galactose by the combined actions of rPcGAL1 and rPcGALX35C. Surprisingly, neither rPcGAL1 nor rPcGALX35C released any galactose from sugar beet pectin. 相似文献
17.
Benjamin Pluvinage Seth Chitayat Elizabeth Ficko-Blean D. Wade Abbott Jobby Maroor Kunjachen Julie Grondin Holly L. Spencer Steven P. Smith Alisdair B. Boraston 《Journal of molecular biology》2013,425(2):334-349
Streptococcus pneumoniae is a serious human pathogen that presents on its surface numerous proteins involved in the host–bacterium interaction. The carbohydrate-active enzymes are particularly well represented among these surface proteins, and many of these are known virulence factors, highlighting the importance of carbohydrate processing by this pathogen. StrH is a surface-attached exo-β-d-N-acetylglucosaminidase that cooperates with the sialidase NanA and the β-galactosidase BgaA to sequentially degrade the nonreducing terminal arms of complex N-linked glycans. This enzyme is a large multi-modular protein that is notable for its tandem N-terminal family GH20 catalytic modules, whose individual X-ray crystal structures were recently reported. StrH also contains C-terminal tandem G5 modules, which are uncharacterized. Here, we report the NMR-determined solution structure of the first G5 module in the tandem, G5-1, which along with the X-ray crystal structures of the GH20 modules was used in conjunction with small-angle X-ray scattering to construct a pseudo-atomic model of full-length StrH. The results reveal a model in which StrH adopts an elongated conformation that may project the catalytic modules away from the surface of the bacterium to a distance of up to ~ 250 Å. 相似文献
18.
《Experimental mycology》1993,17(2):130-141
Awald, P., Zugel, M., Monks, C., Frost, D., and Selitrennikoff, C. P. 1993. Purification of 1,3-β-glucan synthase from Neurospora crassa by product entrapment. Experimental Mycology, 17, 130-141. 1,3-β-Glucan synthase activity of the ascomycete Neurospora crassa was purified ∼700-fold from hyphae. Hyphae were disrupted by bead-beating, and membrane-enriched fractions were obtained by high-speed centrifugation. Membranes were treated with (3-[(3-cholamidopropyl)dimethyl-ammoniol]I-propanesulfonate) and octyl-β-D-glucoside to solubilize enzyme activity. Soluble glucan synthase activity was incubated with substrate (UDP-glucose) and purified by centrifugation of enzyme associated with glucan (product entrapment). Purification was specific for UDP-glucose, the optimal concentration being 0.25 mM; no other nucleotide diphosphate sugar was able to significantly product-entrap enzyme activity. Partially purified enzyme activity formed β(1,3)-linked glucan, had a mean specific activity of 1900 nmol glucose incorporated/min/mg protein, a Km,app of 0.7 mM, and a Vmax of 0.5 nmol glucose incorporated/min. Separation of partially purified enzyme activity by SDS-PAGE showed a number of proteins copurifying with enzyme activity; computer analysis of digitized gel images revealed that proteins of 21, 25, 28, 45, 53, and 78 kDa were enriched. These results reinforce the view that 1,3-β-glucan synthase activity of fungi is a multimeric enzyme. 相似文献
19.
Earl D. Mitchell Becky B. Johnson Tina Whittle 《In vitro cellular & developmental biology. Plant》1980,16(10):907-912
Summary Cotton callus and suspension cultures developed from a cotton variety susceptible toXanthomonas malvacearum (E. F. Sm.) Dow, were grown on chemically defined media that contained one of the carbohydrate sources: 3% sucrose, 3% lactose,
3% maltose, 3% fructose, and 3% glucose. All cells were maintained on a medium with sucrose as the carbohydrate and subsequently
transferred to media containing the above carbohydrates. Sucrose was the best carbon source for a high growth rate; however,
cells growing on glucose, which was almost as good as sucrose, and cells growing on lactose did not turn brown when they reached
the stationary phase of growth. A crude extract from callus tissue growing on lactose has a fivefold increase in β-galactosidase
[EC 3.21.23] activity as compared with the extract from callus tissue growing on sucrose. When callus tissue growing on lactose
was transferred tomedium containing sucrose, β-galactosidase activity decreased to the level as measured in cells maintained
on sucrose. Callus cells growing on a lactose medium showed staining when treated with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside
in which very heavy granular stains appeared. Cells growing on sucrose did not show the histochemical staining. These observations
suggest that β-galactosidase is induced in cotton callus tissue that has been transferred from a medium containing sucrose
to a medium containing lactose.
This is journal article J-3704 of the Oklahoma Agricultural Experiment Station. The research was supported in part by a Presidential
Challenge Grant from Oklahoma State University and the Oklahoma Agricultural Experiment Station. 相似文献
20.
S.V.N. Vijayendra Yutaka KASHIWAGI 《International journal of biological macromolecules》2009,44(1):92-97
A new acid stable exo-β-1,3-glucanase of Rhizoctonia solani purified from a commercial source ‘Kitarase-M’, by a combination of ammonium sulfate precipitation, ion-exchange and gel filtration methods, had specific activity of 0.26 U/mg protein, Km and Vmax values of 0.78 mg/ml and 0.27 mM/min/mg protein, respectively. It had molecular weight of 62 kDa with optimum activity at 40 °C temperature and pH 5.0, with high stability at pH of 3–7. Unique amino acid sequence was found at N-terminal end. The substrate specificity studies confirmed that it is an exo-β-1,3-glucanase. It could hydrolyze curdlan powder to release glucose. 相似文献